Environmental stress in a broad sense is a restriction placed on living organisms by nature. The definition of environmental stress in plant science is a set of physical and chemical factors affecting the environment consequently disturbing plant growth. This stress could occur due to variant temperatures be it high or low, insufficient water supply, ultraviolet radiation and emission of pollutant gases. The study of environmental stress in plant life is significant on account of the fact that it world over agricultural productivity has been greatly restricted by it and the need to withstand this kind of environmental stress is a prerequisite when studying plant life.
Under stressful conditions, the stress factor or toxic molecules derived from the stress factor attack the more sensitive molecules i.e. the primary targets in cells to impair their functions. Cells are protected by the endogenous molecular systems which mitigate the stress. The damaged targets either by having them repaired or replaced are recovered via de novo biosynthesis. When the damage caused by stress to the primary targets is very intense, the cell cannot get over the damage and the metabolic function(s) operated by the target molecules are distorted. If the repair system in respect of the damaged molecules or the energy supply system is impaired, viz, the entire cellular metabolism disintegrates allowing for the propagation of damages, then there is a cascade of events leading to cell death.
Cells have the capability of surviving stressful conditions by sensing stress and adjusting their gene expression pattern to establish new metabolism which adapt to the stress. This adaptive response is known as acclimation and it takes place from a few hours to several days in which time cells take on the stress by making use of pre existing protection systems until the new metabolism is established. The destiny of the cell is determined by the degree of available protection and the intensity and duration of the stress. The investigation of the cellular response in the early stages of environmental stress reveal the endogenous and exogenous factors that determine the stress tolerance of the plant.
The production of reactive oxygen species in cells is an inevitable restriction on aerobic life and use is made of the oxidative atmosphere for yielding energy at a high efficiency. In so far as the metabolism under non stressful conditions is concerned, reactive oxygen species is always produced. The reactive oxygen species is produced in the cells for biosynthesis, cell defence, intra and intercellular signalling. Hence, reactive species of oxygen is both, indispensable as well as toxic to life.
It has been observed that reactive oxygen species plays a crucial role in the impairment of cellular functions due to environmental stress viz, increase in the productive of reactive oxygen species and production of oxidised target molecules under stress, decrease in the antioxidant levels or contents under stress, increased expression of the genes for anti-oxidative functions by stress, positive co-relation between the scavenging capacity for reactive oxygen species and tolerance towards stress, cross tolerance between oxidative stress and other stress.
Oxidative damage caused by reactive oxygen species can be induced by two principal mechanisms viz, an enhanced production of reactive oxygen species or by an inhibition of the scavenging systems for them. The damage proliferates production of a highly reactive hydroxyl radical and the subsequent reactions like bleaching of pigments and accumulation of oxidised lipids are apparent, these being the final symptoms of oxidative damage observed in dying cells.
Superoxide is a commonly encountered mediate of oxygen reduction. It is extremely toxic to cells since it attacks unsaturated fatty acid components of membrane lipids thereby damaging the membrane structure. Aerobic cells detoxify super oxide by the action of super oxide dismutases, metal containing enzymes that convert the superoxide radical into hydrogen peroxide and molecular oxygen. The hydrogen peroxide later converted by catalase into water and molecular oxygen.
There are three types of super oxide dismutase (SOD), copper/zinc containing SOD(CuZnSOD), manganese containing SOD (MnSOD) and iron containing SOD (FeSOD). In prokaryotic organisms MnSOD is inducible under conditions of high oxygen concentration and by O2.
Reactive oxygen species is produced in almost every cell compartment in instances of normal metabolism (Halliwell & Gutteridge, 1989). The chloroplast containing pigments at high concentrations and evolving 02 under light is a major source of reactive species in plant cells under illumination (Asada & Takashahi, 1987). The chloroplast and the leaf tissue is regarded as a primary site of stress induced damage in plants under light. However there are numerous cases wherein the stimuli arise from extrachloroplastic sites, e.g., the ozone, which, penetrates into the leaf tissue and interacts with apoplastic components on account of which the apoplastic antioxidant capacity assumes significance. Biotic stress like bacterial infection and grazing arises from the periphery of the cells. In the case of water stress like drought and high saline content in the soil, stress stimuli are sensed primarily by roots as well as the leaves.
In an instance where the chlorophyll (Ch1) molecule at the photochemical reaction centre in the thylakoid membranes absorbs light energy, a high potential oxidative power i.e. a positive charge and a low potential reducing power, a negative charge is generated. On the oxidative side of the photosystem II (PSII) the oxidative terminus of the photosynthetic electron transport chain, water is oxidised to O2. On the reducing side of photosystem I (PSI) the opposite terminus, the iron-sulphur protein ferredoxin (Fd) is reduced. The reduced Fd provides electrons for CO2 fixation and other reactions in the chloroplast. There are two potential production sites for reactive oxygen species, the reducing side of PSI and PSII.
The redox potential of the FeS centres at the terminus of PSI, 0.4 is low enough to reduce 02 univalently to produce superoxide radical (02). 02+PSI reduced→02+PSI (oxidised). The photoreduction of O2 to O2 by PSI (Asada & Kiso, 1973b) inevitably occur and uses 10-20% of the photosynthetic electron flux even under conditions where C02 supply saturates (Asada & Takahashi, 1987). O2 is disproportionate to H202 and O2 via catalysis by superoxide dismutase (SOD) which is contained in the stroma (Asada et al., 1973).
2O2+2H+→H2O2+O2: These reactions account for most of the photoproduction of H202 in chloroplast (Mehler reaction; Mehler, 1951). H2O2 is produced via non-enzymic reduction of O2 with ascorbate (AsA) or glutathione (GSH).
O2+AH→H2O2+A∀, where AH and A∀ represent either AsA or GSH and its radical, respectively. Under normal physiological conditions this mechanism is neglected since the produced O2 is immediately disprorportioned with SOD, which resides near the production site of O2 (Ogawa et al., 1995).
02 photoproduced from H2O in PSI II is finally reduced to H2O in PSI, with catalysis by SOD and APX, to form a cycle of electron flow (water-water cycle; Asada et. Al, 1998). With regard to the produced reactive oxygen species scavenged in situ by the enzymes of the water cycle, the photoreduction of O2 to O2 is not detrimental but indispensable in preventing photoinhibition of chloroplast by acting as a safety valve that dissipates excessive excitation energy as heat (Schreiber & Neubauer, 1980, Neubauer & Yamamoto, 1922, Osmond & Grace, 1995, Laisk & Edwards, 1998). Even at 1.10/0, C02 that saturates photoreduction of C02 in chloroplast, the electron flow to 02 prevents photoinhibition despite producing 02 (Park et al., 1996). This efficient scavenging of O2 and H2O2 is ensured by high molecular activities and intraorganellar microlocalisation of the water-water cycle enzymes (Asada et al., 1998). The chloroplastic flavoenzyme monodehydroascorbate reductase has been suggested to regulate the photoproduction rate of 02 at PSI (Miyake et al., 1998).
H2O2 is also produced outside the chloroplast not only via the disproportionation of O2 but also via the divalent reduction of O2 catalysed by various oxidases which catalyse divalent oxidation. H202 if provided with reductants and an appropriate catalyst, e.g. transition metal ions, quinones and Fd (Jacob & Heber, 1996), is reduced to form a highly toxic hydroxyl radical (HOψ) (Heber-Weiss reaction). H2O2+AH→HOψ+OH+A. AsA, GSH and 02 can be reductants for this reaction. As catalysts the FeS centres in PSI reaction complex (Sonoike, 1996b) and in Fd (Jacob & Heber, 1996) might produce HOψ in situ. Transition metal ions e.g. Fe, Cu and Mn, if released from metalloenzymes for some reasons are also effective catalysts. Cd from the environment also catalyses the Haber-Weiss reaction. HOψ production is implied in the oxidative stress caused by excess Fe in tobacco (Kampfenkel et al., 1995). HOψ can also be detected on the donor side of PS II which is impaired by UV-B (Hideg & Vass, 1996) although the source and the reaction to produce this radical is not yet known, as of now. HOψ is highly oxidative (redox potential of HOψ/H2O; +2.3 V) and oxidises organic molecules at the constant rate of 109 MD sD1 (Halliwell & Gutteridge, 1989) and is toxic.
At the other end of the electron transport chain, at the time when the charges separated at the Ch1 dimer at the reaction centre recombine, the triplet state of Ch1 (3Ch1) is formed and it reacts rapidly with ground state oxygen (302) to form a singlet oxygen (102). 3Ch1+302→1 Ch1+102. 102 is also produced via a similar photodynamic reaction with heme groups in proteins and with flavins through various reactions from 02D and H2O2 (Halliwell & Gutteridge, 1989). In PSI II reaction centre, 102 is produced when the primary acceptor quinone QA is fully reduced (Vass & Styring, 1993). The photoproduction of 102 in PSI II has been observed in vitro (Macpherson et al., 1993) and in vivo (Hideg et al., 1998). 102 is highly reactive with organic molecules and consequently, highly toxic as well. The oxidative potential generated in the PSII reaction centre required for the oxidation of water to oxygen is potentially toxic to the PSII complex itself and damages it as a probable event (Anderson et al., 1998). The oxidant is harnessed with a charge accumulation mechanism of the Mn cluster of water oxidase (Kok et al., 1970) so as not to release the possibly generated intermediates of water oxidation, HOψ, H202 and 02D. When water oxidase is destroyed on account of some reason or the other, such as UV-B or heat, the photogenerated oxidative power as P680+ or Tyrz+, may, oxidise the surrounding protein matrix or neighbouring molecules to inactivate PSII complex (donor-side-induced photoinhibition; Blubaugh et al., 1991, Aro et al, 1993). Further, reactive oxygen species that can be produced through photooxidation of water, may be released (Ananyev et al., 1992, Fine & Frasch, 1992, Hideg et al., 1994).
In additional chloroplastic compartments, the major production reaction for reactive oxygen species are not only the univalent reduction of 02 to 02D but the divalent reduction of 02 to H202. Peroxisomes contain divalent reaction oxidases and produce H2O2 in association with oxidative metabolisms like photorespiration and -oxidation of lipids. In C3 plants a substantial amount of H202 is produced and accompanies the photorespiration through the peroxisomal glycolate oxidase.O2+glycolate→H2O2+glycolate.
Acyl-CoA oxidase in peroxidase catalyses divalent oxidation of acyl-CoA to trans-2, 3-dehydroacyl-CoA by O2 in the beta-oxidation of lipids, producing H202. O2D is produced in mitochondria. In mammalian mitochondria, O2D production due to electron leakage from the electron transport to O2 accounts for 1-2% of total electron flux through the chain (Chance et al., 1979) and is increased several fold by the inhibitors of electron transport, uncouplers and other agents to disrupt mitochondrial functions (Richter & Schweizer, 1997). The production of O2D in submitochondrial particles from pea leaves has been demonstrated (Hernandez et al., 1993). Assuming that mitochondria is a major production site of O2D in non photosynthetic cells, it has not yet been elucidated as to whether the production of O2D in mitochondria has a physiologically positive significance as that in the chloroplast. O2D is also produced in peroxisome and plasma membrane. In plant peroxisome, O2D is produced via xanthine oxidase and at least three distinct NAD (P)H oxidases (del Rio, 1998). Peroxisomal O2D production is increased during senescence and the reactive oxygen species derived from it, decompose cellular components (Brennan & Frenkel, 1977, del Rio et al., 1998). Participation in the production of O2D of a mammalian like NADPH-oxidase on the plasma membrane in plant cells has been established upon extracellular stimuli (Auh & Murphy, 1995, Allan & Fluhr, 1997) and during lignification (Ogawa et al., 1997).
Reactive oxygen species have their respective molecular properties and reactivities with biomolecules with scavenging mechanisms for both. O2D is generally known as a relatively stable or unreactive molecule among the reactive oxygen species. However the protonated form H02 (pKa=4.8) is a much higher reactive. H02 can initiate lipid peroxidation but not O2D. Moreover HO2 can pass across lipid bilayers but not O2D. In an aqueous solution, O2D spontaneously disproportionates to form H2O2 and O2.O2D+O2D+2H+→H2O2+O2 
At a lower pH, the following reactions may occur:O2D+HO2+H+H2O2+O2 HO2+HO2→H2O2+O2 
The second order rate constants for these reactions are <0.35 MD1 sD1, 1.02×107 MD1 sD1 and 8.60×105 MD1 sD1. Since the reaction constant is the largest apparent second order rate constant for the disproportionation of O2D, 5-105 MD1 sD1 at pH 7.0, thus decreases by 10 fold per each pH unit increase in the range over pH 5 (Bielski, 1978). O2D is a reductant of the transition metal ions in the Haber-Weiss reaction to produce HOψ from H2O2. O2D also propagates radical chain reaction especially in the presence of quinone. When quniones are univalently reduced to semiquinones (QHψ) with quinone reductases which abundantly occur in plant cells, parts of the QHψ reduces dioxygen to produce O2D, which oxidises the quinols that have been produced via the disproportination of QHψ to reproduce QHψ. This chain reaction is effectively terminated by SOD (Cadenas et al., 1992).
O2D is highly reactive with reduced sulfur compounds like thiols and FeS cluster. O2D oxidises thiols to the thiyl radicals at diffusion controlled rates (Asada & Kanematsu, 1976). The resulting thiyl radicals initiate radical chain reaction. O2D also oxidises the 4 Fe-4S) cluster of aconitase in mammalian mitochondria or in bacteria at the order of 106-107 MD1 sD1 to the inactive (3Fe-4S) form (Radi et al., 1998). The Fe2+ ion released as a consequence is a potent catalyst for Haber-Weiss reaction. In plant cells the major SOD isozymes are located in chloroplasts (MnSOD). The occurrence of CuZnSOD in the apoplast and nucleus has been confirmed by immunoelectron microscopy (Ogawa et al., 1995). The occurrence of SOD implies the in situ production of O2D. CuZnSOD and FeSOD are sensitive to H2O2. These SODS are the potential targets if the H2O2 scavenging systems do not operate properly.
H2O2 is a neutral, non radical molecule below pH 10 and can diffuse across biomembranes like water. The function of H2O2 as a stress signal (Doke, 1997) is partly based on its intra and inter cellular diffusability. H2O2 is a relatively weak oxidant, the oxidative potential of H2O2/H2O pair is +320 mV. However, metalloenzymes are in general sensitive targets of H2O2. Heme proteins can catalyse the Haber-Weiss reaction and can be degraded by the resulting HOψ (Puppo & Halliwell, 1998). Chloroplastic APX isozymes are inactivated by H2O2 in the absence of electron donors (Hossain & Asada, 1984) since compound I is irreversibly oxidised by H202 (Miyake & Asada, 1996). CuZnSOD is inactivated by H2O2 (Bray et al., 1974) through the reduction of Cu2+ ion at the reaction centre to Cu+ and the subsequent production of HOψ (Hodgson & Fridovich, 1975). CuZnSOD in isolated chloroplast of wheat leaves are inactivated by insufficient light probably due to photoproduced H2O2 (Casano et al., 1997). FeSOD is also inactivated by H2O2 (Beyer & Fridovich, 1987). The inactivation of these enzymes have been observed in vitro at the _M to sub-mm range of H2O2, which can be reached in vivo as well if the H2O2 scavenging systems do not operate effectively. H2O oxidises thiols to the sulfenic acids which react with thiols to form disulfides. The reaction between H2O2 and cysteine is slow (the apparent second order rate constant, 1MD1, sD1) but on the surface of the proteins the reaction may be largely accelerated by the presence of basic residue like Lys and Arg which could be the neighbouring thiol groups (Armstrong & Buchanan, 1978). H2O2 at micromolar concentrations in darkness inhibit C02 fixation in the chloroplast by 50% in 10 min (Kaiser, 1979) due to the oxidation of the active site thiols to the disulfide in the Calvin cycle enzymes; fructose-1; biphosphatase, NADP-glyceraldehydes-3-phosphate dehydrogenase and ribulose-5 phosphate kinase. The activities of these inhibited enzymes are recovered by the reduction with reduced thioredoxin reversibly (Wolosiuk & Buchanan, 1977). However, if cessation of CO2 continues under light, it will lead to excess light energy wherein the production of reactive oxygen species increases.
H2O2 is scavenged by two types of enzymes, catalase and peroxidase. The former scavenges H202 through the disproportination of H2O2 to O2 and H2O corresponding to a turnover rate of about 107 minD1. (Scandalios et al., 1997). 2H2O2—O2+2H2O. Plants have several catalase isozymes, which are expressed in the regulated stage and tissue (Scandalios et al., 1997). Catalase is localised mainly in peroxisomes and responsible for scavenging the H2O2 produced in photorespiration and beta-oxidation of lipids. Catalase is a key antioxidant enzyme, a tetrameric heme containing enyme found in nearly all the aerobic organisms which converts hydrogen peroxide into water and molecular oxygen in plants and are primarily located in peroxisomes. Plant catalases are involved in the detoxification of active oxygen species which are generated during the course of photorespiration, the beta-oxidation of fatty acids or different environmental stresses (Scandalios, 1990).
It has been shown that induction of superoxide dismutase activity in plant cells has been correlated with development of increased tolerance to a variety of chemical compounds and physical stress. Environmental stress is known to decrease crop activity according to the severity and type of stress. Enhancing tolerance of crop plants to adverse effects imposed by non optimal growing conditions for improvement of crop management. There is hence, a substantial interest in the ability to increase the concentration of super oxide dismutase in a plant cell so as to provide for a plant which has increased tolerance to environmental stress.